Title: Enhanced Apoptosis of B and T Lymphocytes in TAFII105 Dominant-negative Transgenic Mice Is Linked to Nuclear Factor-κB
Abstract: The general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 12–14 TBP-associated factors (TAFIIs). Some TAFIIs act as bridges between transcription activators and the general transcription machinery through direct interaction with activation domains. Although TAF-mediated transcription activation has been established, there is little genetic evidence connecting it to binding of an activator. TAFII105 is a substoichiometric subunit of transcription factor IID highly expressed in B lymphocytes. In this study, we examined the physiological role of TAFII105 and its mechanism of action in vivo by expressing two forms of dominant-negative mutant TAFII105 in mice. We show that TAFII105 has a pro-survival role in B and T lymphocytes, where the native protein is expressed. In addition, TAFII105 is important for T cell maturation and for production of certain antibody isotypes. These phenotypic alterations were absent in mice expressing a dominant-negative mutant that lacks one of the domains mediating p65/RelA binding in vitro. These findings provide support to the notion that interaction between the activator and TAF is important for their function in vivo. The general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 12–14 TBP-associated factors (TAFIIs). Some TAFIIs act as bridges between transcription activators and the general transcription machinery through direct interaction with activation domains. Although TAF-mediated transcription activation has been established, there is little genetic evidence connecting it to binding of an activator. TAFII105 is a substoichiometric subunit of transcription factor IID highly expressed in B lymphocytes. In this study, we examined the physiological role of TAFII105 and its mechanism of action in vivo by expressing two forms of dominant-negative mutant TAFII105 in mice. We show that TAFII105 has a pro-survival role in B and T lymphocytes, where the native protein is expressed. In addition, TAFII105 is important for T cell maturation and for production of certain antibody isotypes. These phenotypic alterations were absent in mice expressing a dominant-negative mutant that lacks one of the domains mediating p65/RelA binding in vitro. These findings provide support to the notion that interaction between the activator and TAF is important for their function in vivo. The basal transcription factor TFIID is a multisubunit complex consisting of TATA-binding protein (TBP) 1The abbreviations used are: TBPTATA-binding proteinTAFIITFIID-associated factorTAFTBP-associated factorNF-κBnuclear factor-κBKLHkeyhole limpet hemocyaninRT-PCRreverse transcription-polymerase chain reactionGAPDHglyceraldehyde-3-phosphate dehydrogenaseFACSfluorescence-activated cell sorter 1The abbreviations used are: TBPTATA-binding proteinTAFIITFIID-associated factorTAFTBP-associated factorNF-κBnuclear factor-κBKLHkeyhole limpet hemocyaninRT-PCRreverse transcription-polymerase chain reactionGAPDHglyceraldehyde-3-phosphate dehydrogenaseFACSfluorescence-activated cell sorter and associated factors (TAFIIs). TAFIIs display multiple functions related to transcription regulation (1Hoffmann A. Oelgeschlager T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8928-8935Crossref PubMed Scopus (76) Google Scholar, 2Verrijzer C.P. Tjian R. Genes Dev. 1996; 11: 3020-3031Google Scholar). They are required for recognition and binding to core promoter elements such as the initiator and the downstream promoter element (3Deleted in proofGoogle Scholar, 4Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (176) Google Scholar, 5Kauffmann L. Smale S. Genes Dev. 1994; 8: 821-829Crossref PubMed Scopus (213) Google Scholar, 6Martinez E. Chiang C.-M., Ge, H. Roeder R.G. EMBO J. 1994; 13: 3115-3126Crossref PubMed Scopus (159) Google Scholar, 7Smale S.T. Biochim. Biophys. Acta. 1997; 1351: 73-88Crossref PubMed Scopus (499) Google Scholar, 8Verrijzer C.P.,Y.K. Chen J.L. Tjian R. Science. 1994; 264 (Y. K.): 933-941Crossref PubMed Scopus (178) Google Scholar). In addition, certain TAFIIs interact with activation domains of transcription factors, interactions that were found to be essential for transcription activation by activators in vitro (9Albright S.R. Tjian R. Gene (Amst.). 2000; 242: 1-13Crossref PubMed Scopus (270) Google Scholar). The importance of TAFIIs in the transcription activation process has also been supported by several studies in cultured cells (10Bertolotti A. Bell B. Tora L. Oncogene. 1999; 18: 8000-8010Crossref PubMed Scopus (69) Google Scholar, 11Lavigne A.C. Mengus G. Gangloff Y.G. Wurtz J.M. Davidson I. Mol. Cell. Biol. 1999; 19: 5486-5494Crossref PubMed Google Scholar, 12May M. Mengus G. Lavigne A.C. Chambon P. Davidson I. EMBO J. 1996; 15: 3093-3104Crossref PubMed Scopus (75) Google Scholar, 13Mengus G. May M. Carre L. Chambon P. Davidson I. Genes Dev. 1997; 11: 1381-1395Crossref PubMed Scopus (132) Google Scholar, 14Wolstein O. Silkov A. Revach M. Dikstein R. J. Bio. Chem. 2000; 275: 16459-16465Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 15Yamit-Hezi A. Dikstein R. EMBO J. 1998; 17: 5161-5169Crossref PubMed Scopus (68) Google Scholar, 16Deleted in proofGoogle Scholar) and by genetic experiments in Drosophila (17Pham A.D. Muller S. Sauer F. Mech. Dev. 1999; 84: 3-16Crossref PubMed Scopus (22) Google Scholar,18Zhou J. Zwicker J. Szymanski P. Levine M. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13483-13488Crossref PubMed Scopus (60) Google Scholar).Despite the significant progress that has been made in the characterization of TAFIIs at the biochemical level, little is known about the physiological relevance of these studies or about the specific functions of individual TAF subunits in biological processes involving transcription regulatory programs. An important issue concerning the mechanism of TAF-mediated transcription activation is whether direct contact between activators and TAFIIs is indeed essential for transcription in vivo and what is the consequence of activator-TAF interaction in vivo. Examination of activator-TAF connection is of particular interest because activators have multiple potential coactivator targets within the transcription machinery.Studies in yeast carrying mutations in individual TAFIIsubunits indicated that some TAFIIs are required for transcription of the majority of class II genes and others for transcription activation of only subsets of genes (19Apone L.M. Virbasius C.M. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (129) Google Scholar, 20Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Metzger D. Scheer E. Soldatov A. Tora L. EMBO J. 1999; 18: 4823-4834Crossref PubMed Scopus (75) Google Scholar, 22Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (250) Google Scholar, 24Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 25Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Crossref PubMed Scopus (52) Google Scholar, 27Sanders S.L. Klebalow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar). Interestingly, inactivation of certain individual TAFIIs dramatically affects the stability and integrity of the entire TFIID complex in vivo (5Kauffmann L. Smale S. Genes Dev. 1994; 8: 821-829Crossref PubMed Scopus (213) Google Scholar, 20Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 24Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 27Sanders S.L. Klebalow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar, 29Reese J.C. Zhang Z. Kurpad H. J. Biol. Chem. 2000; 275: 17391-17398Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), complicating the understanding of the function of the mutant subunit. Furthermore, some of the TAFIIs are shared between TFIID and other complexes such as yeast SAGA and human PCAF and TFTC (30Grant P.A. Schielz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates R., Jr. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 31Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar); thus, a phenotypic alteration observed with these mutant TAFIIs may be linked to any of the complexes containing TAF.TAFII105 is a member of the human TAFII135 and DrosophilaTAFII110 family of TAFIIs, which has several unique properties. Unlike the core TAFIIs that are expressed in most cell lines at similar levels, TAFII105 expression is regulated. It is more abundant in the TFIID complex of human B lymphocytes than in non-B cells (32Dikstein R. Zhou S. Tjian R. Cell. 1996; 87: 137-146Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Consistent with this expression pattern, TAFII105 was found to be involved in transcription activation by p65/RelA, a member of the NF-κB family (15Yamit-Hezi A. Dikstein R. EMBO J. 1998; 17: 5161-5169Crossref PubMed Scopus (68) Google Scholar), and OCA-B (14Wolstein O. Silkov A. Revach M. Dikstein R. J. Bio. Chem. 2000; 275: 16459-16465Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar); both are required for lymphocytic gene expression. TAFII105 appears to be present only in a small fraction of TFIID complexes and therefore might be involved in transcription of a relatively small subset of genes. The C-terminal domain of human TAFII105 and TAFII130 and Drosophila TAFII110 is highly conserved and has interaction surfaces with other TAFIIs, implicating it in the assembly of the TFIID complex. The N terminus of TAFII105 is more variable and contains binding sites for the activation domains of p65/RelA and OCA-B (14Wolstein O. Silkov A. Revach M. Dikstein R. J. Bio. Chem. 2000; 275: 16459-16465Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 15Yamit-Hezi A. Dikstein R. EMBO J. 1998; 17: 5161-5169Crossref PubMed Scopus (68) Google Scholar). Because TAFII105 is found only in a small portion of TFIID complexes and may be required for transcription of a small subset of genes, but is homologous to one of the core subunits, it has the potential to be a good candidate for genetic analysis in mammals. However, recent data obtained from the Human Genome Project revealed another gene on chromosome 4 encoding a close homolog of TAFII105 (accession number AC017007). The previously identified gene is encoded by chromosome 18 (accession number Y09321). The existence of an expressed sequence tag clone identical to the chromosome 4 gene and the fact that a sequence of one of the peptides derived from the TAFII105 protein more closely resembles the chromosome 4 gene (data not shown) strongly suggest that this gene is also expressed and may display some redundant functions with the gene encoded by chromosome 18. Recently, mice deficient in TAFII105 were generated; the females of these mice were found to be sterile due to defects in folliculogenesis, but no obvious phenotypic alterations in lymphoid organs were found (33Freiman R.N. Albright S.R. Zheng S. Sha W., C. Hammer R.E. Tjian R. Science. 2001; 293: 2084-2087Crossref PubMed Scopus (164) Google Scholar).In this study, we addressed the physiological role and the mechanism of action of TAFII105 in vivo by generating animal models expressing dominant-negative mutant forms of TAFII105 that are likely to inhibit the function of TAFII105 proteins encoded by different genes. The results revealed that TAFII105 is essential for the survival of B and T lymphocytes, where the native protein is highly expressed. This function of TAFII105 is dependent on a domain involved in the interaction with the NF-κB protein p65/RelA in vitro, suggesting that activator-TAF interaction is important for the function of TAFIIs in the transcription process in vivo.DISCUSSIONIn this study, we have examined the function of TAFII105 in the context of the whole organism and determined the biological importance of a domain in TAFII105 involved in interaction with the NF-κB protein p65/RelA in vitro. We generated and analyzed transgenic mice expressing dominant-negative mutants of TAFII105, which inhibit the function of TAFII105 and, most likely, also of TAFII105 homologs. Because we did not interfere with expression of the native protein, this strategy did not affect the stability of the endogenous TAFII105·TFIID complex. Furthermore, expression of different forms of dominant-negative mutants allowed us to examine the structure-function relationship involved in the mechanism of action of TAFII105 in vivo. We provide evidence that one of the domains within TAFII105 required for interaction with p65/RelA in vitro is crucial for lymphocyte survival and antibody production in the mouse. This notion is consistent with previous findings indicating that TAFII105ΔC-ΔNFκB is a significantly less potent inhibitor of NF-κB transcription activity than TAFII105ΔC (35Yamit-Hezi A. Nir S. Wolstein O. Dikstein R. J. Biol. Chem. 2000; 275: 18180-18187Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and strongly suggests that the C-terminal NF-κB-binding site within TAFII105 is a functionally more important site than the N-terminal site. As NF-κB has been established as a survival factor in lymphocytes (39Barkett M. Gilmore T. Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1066) Google Scholar, 40de Martin R. Schmidt J.A. Hofer-Warbinek R. Mutat. Res. 1999; 437: 231-243Crossref PubMed Scopus (71) Google Scholar, 41Foo S.Y. Nolan G.P. Trends Genet. 1999; 15: 229-235Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 42Grossmann M. O'Reilly L.A. Gugasyan R. Strasser A. Adams J.M. Gerondakis S. EMBO J. 2000; 19: 6351-6360Crossref PubMed Scopus (192) Google Scholar, 43Huesmann M. Scott B. Kieslow P. von Boehmer H. Cell. 1991; 66: 533-542Abstract Full Text PDF PubMed Scopus (321) Google Scholar), the finding that TAFII105 is important for activation of anti-apoptotic genes in B and T lymphocytes implies that it cooperates with p65/RelA in these cells to activate some of the NF-κB target genes. The observation that the NF-κB interaction domain is also required for IgM, IgG2a, and IgG3 isotype switching is also consistent with a recent finding showing that B cells lacking p65 exhibit a defect in switching to IgG3 (44Horwitz B.H. Zelazowski P. Shen Y. Wolcott K.M. Scott M.L. Baltimore D. Snapper C.M. J. Immunol. 1999; 15: 1941-1946Google Scholar). However, it has yet to be determined whether the effect of TAFII105ΔC expression on lymphocyte survival is intrinsic or involves contributions of other cell types.Programmed cell death is of fundamental importance in the immune system and plays important roles in the control of the immune response and in lymphocyte development and cytotoxicity (reviewed in Refs. 45Cohen J.J. Duke R.C. Fadok V.A. Sellins K.S. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1102) Google Scholar, 46Williams G.T. Biochem. Cell Biol. 1994; 72: 447-450Crossref PubMed Scopus (13) Google Scholar, 47Wyllie A.H. Int. Rev. Cytol. 1987; 17: 755-785Google Scholar). In developing lymphocytes, cell death is the mechanism by which immune cells that recognize self-antigens are deleted. This process ensures the release of cells recognizing non-self-antigens into the periphery and determines the finite life span of terminally differentiated cells. Thus, apoptosis provides a flexible mechanism for controlling the composition and size of the mature cell population. It has been proposed that positive selection of thymocytes is a rescue from a default pathway of death in developing thymocytes (48Egerton M. Scollay R. Shortman K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2579-2582Crossref PubMed Scopus (279) Google Scholar, 49Deleted in proofGoogle Scholar, 50Surh C.D. Sprent J. Nature. 1994; 372: 100-103Crossref PubMed Scopus (930) Google Scholar). The TAFII105·NF-κB complex may be required for the positive selection process during lymphocyte development and the immune response by translating upstream positive signals into activation of survival gene products. This idea is supported by our observation of a significant reduction in the relative numbers of single-positive mature T cells in TAFII105ΔC transgenic mice. This effect may be explained in part by the increased rate of apoptosis in the population of immature double-positive thymocytes, from which mature T cells develop by positive selection. However, given that the proportion of the immature double-positive T cells in the thymuses of TAFII105ΔC mice is not decreased despite high apoptotic rates, it is possible that TAFII105 is important for T cell maturation processes that are also apoptosis-independent.The transgenic animals that we have obtained expressed very low amounts of the dominant-negative proteins relative to the native endogenous protein. The accelerated apoptosis of lymphocytes observed despite low expression levels of the TAFII105ΔC transgene may result from the fact that the decision of lymphocytes to undergo apoptosis is dependent on the extent of positive and negative survival signals encountered by the cell. The amount of TAFII105ΔC protein in the lymphocytes of the transgenic animals may be sufficient to shift a delicate balance of pro- and anti-apoptotic signals toward apoptosis. Alternatively, cells expressing higher levels of the transgene are those that may have been eliminated by apoptosis. It is therefore likely that TAFII105 has additional functions that were not explored in this study, as inhibition of other functions of TAFII105 may require expression levels that are at least comparable to those of the native protein. Our inability to obtain transgenic animals expressing high levels of this protein and the decline of its expression over generations raise the possibility that the protein may be toxic to the animals.Early biochemical studies have suggested that activator-TFIID interaction enhances the formation of a functional preinitiation complex (51Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 52Orlando V. Strutt H. Paro R. Methods: A Companion to Methods in Enzymology. 11. Academic Press, Inc., San Diego, CA1997: 205-214Google Scholar, 53Roeder R.G. Trends Biochem. Sci. 1991; 16: 402-408Abstract Full Text PDF PubMed Scopus (346) Google Scholar). A question arising from this study is whether p65/RelA-TAFII105 interaction contributes to the assembly of the preinitiation complex by enhancing recruitment of the substoichiometric TAFII105·TFIID complex to the promoters of anti-apoptotic genes expressed in lymphocytes. Future experiments should address the molecular mechanism by which NF-κB and its coactivators affect the transcription process in vivo. The basal transcription factor TFIID is a multisubunit complex consisting of TATA-binding protein (TBP) 1The abbreviations used are: TBPTATA-binding proteinTAFIITFIID-associated factorTAFTBP-associated factorNF-κBnuclear factor-κBKLHkeyhole limpet hemocyaninRT-PCRreverse transcription-polymerase chain reactionGAPDHglyceraldehyde-3-phosphate dehydrogenaseFACSfluorescence-activated cell sorter 1The abbreviations used are: TBPTATA-binding proteinTAFIITFIID-associated factorTAFTBP-associated factorNF-κBnuclear factor-κBKLHkeyhole limpet hemocyaninRT-PCRreverse transcription-polymerase chain reactionGAPDHglyceraldehyde-3-phosphate dehydrogenaseFACSfluorescence-activated cell sorter and associated factors (TAFIIs). TAFIIs display multiple functions related to transcription regulation (1Hoffmann A. Oelgeschlager T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8928-8935Crossref PubMed Scopus (76) Google Scholar, 2Verrijzer C.P. Tjian R. Genes Dev. 1996; 11: 3020-3031Google Scholar). They are required for recognition and binding to core promoter elements such as the initiator and the downstream promoter element (3Deleted in proofGoogle Scholar, 4Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (176) Google Scholar, 5Kauffmann L. Smale S. Genes Dev. 1994; 8: 821-829Crossref PubMed Scopus (213) Google Scholar, 6Martinez E. Chiang C.-M., Ge, H. Roeder R.G. EMBO J. 1994; 13: 3115-3126Crossref PubMed Scopus (159) Google Scholar, 7Smale S.T. Biochim. Biophys. Acta. 1997; 1351: 73-88Crossref PubMed Scopus (499) Google Scholar, 8Verrijzer C.P.,Y.K. Chen J.L. Tjian R. Science. 1994; 264 (Y. K.): 933-941Crossref PubMed Scopus (178) Google Scholar). In addition, certain TAFIIs interact with activation domains of transcription factors, interactions that were found to be essential for transcription activation by activators in vitro (9Albright S.R. Tjian R. Gene (Amst.). 2000; 242: 1-13Crossref PubMed Scopus (270) Google Scholar). The importance of TAFIIs in the transcription activation process has also been supported by several studies in cultured cells (10Bertolotti A. Bell B. Tora L. Oncogene. 1999; 18: 8000-8010Crossref PubMed Scopus (69) Google Scholar, 11Lavigne A.C. Mengus G. Gangloff Y.G. Wurtz J.M. Davidson I. Mol. Cell. Biol. 1999; 19: 5486-5494Crossref PubMed Google Scholar, 12May M. Mengus G. Lavigne A.C. Chambon P. Davidson I. EMBO J. 1996; 15: 3093-3104Crossref PubMed Scopus (75) Google Scholar, 13Mengus G. May M. Carre L. Chambon P. Davidson I. Genes Dev. 1997; 11: 1381-1395Crossref PubMed Scopus (132) Google Scholar, 14Wolstein O. Silkov A. Revach M. Dikstein R. J. Bio. Chem. 2000; 275: 16459-16465Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 15Yamit-Hezi A. Dikstein R. EMBO J. 1998; 17: 5161-5169Crossref PubMed Scopus (68) Google Scholar, 16Deleted in proofGoogle Scholar) and by genetic experiments in Drosophila (17Pham A.D. Muller S. Sauer F. Mech. Dev. 1999; 84: 3-16Crossref PubMed Scopus (22) Google Scholar,18Zhou J. Zwicker J. Szymanski P. Levine M. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13483-13488Crossref PubMed Scopus (60) Google Scholar). TATA-binding protein TFIID-associated factor TBP-associated factor nuclear factor-κB keyhole limpet hemocyanin reverse transcription-polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase fluorescence-activated cell sorter TATA-binding protein TFIID-associated factor TBP-associated factor nuclear factor-κB keyhole limpet hemocyanin reverse transcription-polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase fluorescence-activated cell sorter Despite the significant progress that has been made in the characterization of TAFIIs at the biochemical level, little is known about the physiological relevance of these studies or about the specific functions of individual TAF subunits in biological processes involving transcription regulatory programs. An important issue concerning the mechanism of TAF-mediated transcription activation is whether direct contact between activators and TAFIIs is indeed essential for transcription in vivo and what is the consequence of activator-TAF interaction in vivo. Examination of activator-TAF connection is of particular interest because activators have multiple potential coactivator targets within the transcription machinery. Studies in yeast carrying mutations in individual TAFIIsubunits indicated that some TAFIIs are required for transcription of the majority of class II genes and others for transcription activation of only subsets of genes (19Apone L.M. Virbasius C.M. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (129) Google Scholar, 20Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Metzger D. Scheer E. Soldatov A. Tora L. EMBO J. 1999; 18: 4823-4834Crossref PubMed Scopus (75) Google Scholar, 22Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (250) Google Scholar, 24Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 25Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Crossref PubMed Scopus (52) Google Scholar, 27Sanders S.L. Klebalow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar). Interestingly, inactivation of certain individual TAFIIs dramatically affects the stability and integrity of the entire TFIID complex in vivo (5Kauffmann L. Smale S. Genes Dev. 1994; 8: 821-829Crossref PubMed Scopus (213) Google Scholar, 20Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 24Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 27Sanders S.L. Klebalow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar, 29Reese J.C. Zhang Z. Kurpad H. J. Biol. Chem. 2000; 275: 17391-17398Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), complicating the understanding of the function of the mutant subunit. Furthermore, some of the TAFIIs are shared between TFIID and other complexes such as yeast SAGA and human PCAF and TFTC (30Grant P.A. Schielz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates R., Jr. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 31Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar); thus, a phenotypic alteration observed with these mutant TAFIIs may be linked to any of the complexes containing TAF. TAFII105 is a member of the human TAFII135 and DrosophilaTAFII110 family of TAFIIs, which has several unique properties. Unlike the core TAFIIs that are expressed in most cell lines at similar levels, TAFII105 expression is regulated. It is more abundant in the TFIID complex of human B lymphocytes than in non-B cells (32Dikstein R. Zhou S. Tjian R. Cell. 1996; 87: 137-146Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Consistent with this expression pattern, TAFII105 was found to be involved in transcription activation by p65/RelA, a member of the NF-κB family (15Yamit-Hezi A. Dikstein R. EMBO J. 1998; 17: 5161-5169Crossref PubMed Scopus (68) Google Scholar), and OCA-B (14Wolstein O. Silkov A. Revach M. Dikstein R. J. Bio. Chem. 2000; 275: 16459-16465Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar); both are required for lymphocytic gene expression. TAFII105 appears to be present only in a small fraction of TFIID complexes and therefore might be involved in transcription of a relatively small subset of genes. The C-terminal domain of human TAFII105 and TAFII130 and Drosophila TAFII110 is highly conserved and has interaction surfaces with other TAFIIs, implicating it in the assembly of the TFIID complex. The N terminus of TAFII105 is more variable and contains binding sites for the activation domains of p65/RelA and OCA-B (14Wolstein O. Silkov A. Revach M. Dikstein R. J. Bio. Chem. 2000; 275: 16459-16465Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 15Yamit-Hezi A. Dikstein R. EMBO J. 1998; 17: 5161-5169Crossref PubMed Scopus (68) Google Scholar). Because TAFII105 is found only in a small portion of TFIID complexes and may be required for transcription of a small subset of genes, but is homologous to one of the core subunits, it has the potential to be a good candidate for genetic analysis in mammals. However, recent data obtained from the Human Genome Project revealed another gene on chromosome 4 encoding a close homolog of TAFII105 (accession number AC017007). The previously identified gene is encoded by chromosome 18 (accession number Y09321). The existence of an expressed sequence tag clone identical to the chromosome 4 gene and the fact that a sequence of one of the peptides derived from the TAFII105 protein more closely resembles the chromosome 4 gene (data not shown) strongly suggest that this gene is also expressed and may display some redundant functions with the gene encoded by chromosome 18. Recently, mice deficient in TAFII105 were generated; the females of these mice were found to be sterile due to defects in folliculogenesis, but no obvious phenotypic alterations in lymphoid organs were found (33Freiman R.N. Albright S.R. Zheng S. Sha W., C. Hammer R.E. Tjian R. Science. 2001; 293: 2084-2087Crossref PubMed Scopus (164) Google Scholar). In this study, we addressed the physiological role and the mechanism of action of TAFII105 in vivo by generating animal models expressing dominant-negative mutant forms of TAFII105 that are likely to inhibit the function of TAFII105 proteins encoded by different genes. The results revealed that TAFII105 is essential for the survival of B and T lymphocytes, where the native protein is highly expressed. This function of TAFII105 is dependent on a domain involved in the interaction with the NF-κB protein p65/RelA in vitro, suggesting that activator-TAF interaction is important for the function of TAFIIs in the transcription process in vivo. DISCUSSIONIn this study, we have examined the function of TAFII105 in the context of the whole organism and determined the biological importance of a domain in TAFII105 involved in interaction with the NF-κB protein p65/RelA in vitro. We generated and analyzed transgenic mice expressing dominant-negative mutants of TAFII105, which inhibit the function of TAFII105 and, most likely, also of TAFII105 homologs. Because we did not interfere with expression of the native protein, this strategy did not affect the stability of the endogenous TAFII105·TFIID complex. Furthermore, expression of different forms of dominant-negative mutants allowed us to examine the structure-function relationship involved in the mechanism of action of TAFII105 in vivo. We provide evidence that one of the domains within TAFII105 required for interaction with p65/RelA in vitro is crucial for lymphocyte survival and antibody production in the mouse. This notion is consistent with previous findings indicating that TAFII105ΔC-ΔNFκB is a significantly less potent inhibitor of NF-κB transcription activity than TAFII105ΔC (35Yamit-Hezi A. Nir S. Wolstein O. Dikstein R. J. Biol. Chem. 2000; 275: 18180-18187Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and strongly suggests that the C-terminal NF-κB-binding site within TAFII105 is a functionally more important site than the N-terminal site. As NF-κB has been established as a survival factor in lymphocytes (39Barkett M. Gilmore T. Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1066) Google Scholar, 40de Martin R. Schmidt J.A. Hofer-Warbinek R. Mutat. Res. 1999; 437: 231-243Crossref PubMed Scopus (71) Google Scholar, 41Foo S.Y. Nolan G.P. Trends Genet. 1999; 15: 229-235Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 42Grossmann M. O'Reilly L.A. Gugasyan R. Strasser A. Adams J.M. Gerondakis S. EMBO J. 2000; 19: 6351-6360Crossref PubMed Scopus (192) Google Scholar, 43Huesmann M. Scott B. Kieslow P. von Boehmer H. Cell. 1991; 66: 533-542Abstract Full Text PDF PubMed Scopus (321) Google Scholar), the finding that TAFII105 is important for activation of anti-apoptotic genes in B and T lymphocytes implies that it cooperates with p65/RelA in these cells to activate some of the NF-κB target genes. The observation that the NF-κB interaction domain is also required for IgM, IgG2a, and IgG3 isotype switching is also consistent with a recent finding showing that B cells lacking p65 exhibit a defect in switching to IgG3 (44Horwitz B.H. Zelazowski P. Shen Y. Wolcott K.M. Scott M.L. Baltimore D. Snapper C.M. J. Immunol. 1999; 15: 1941-1946Google Scholar). However, it has yet to be determined whether the effect of TAFII105ΔC expression on lymphocyte survival is intrinsic or involves contributions of other cell types.Programmed cell death is of fundamental importance in the immune system and plays important roles in the control of the immune response and in lymphocyte development and cytotoxicity (reviewed in Refs. 45Cohen J.J. Duke R.C. Fadok V.A. Sellins K.S. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1102) Google Scholar, 46Williams G.T. Biochem. Cell Biol. 1994; 72: 447-450Crossref PubMed Scopus (13) Google Scholar, 47Wyllie A.H. Int. Rev. Cytol. 1987; 17: 755-785Google Scholar). In developing lymphocytes, cell death is the mechanism by which immune cells that recognize self-antigens are deleted. This process ensures the release of cells recognizing non-self-antigens into the periphery and determines the finite life span of terminally differentiated cells. Thus, apoptosis provides a flexible mechanism for controlling the composition and size of the mature cell population. It has been proposed that positive selection of thymocytes is a rescue from a default pathway of death in developing thymocytes (48Egerton M. Scollay R. Shortman K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2579-2582Crossref PubMed Scopus (279) Google Scholar, 49Deleted in proofGoogle Scholar, 50Surh C.D. Sprent J. Nature. 1994; 372: 100-103Crossref PubMed Scopus (930) Google Scholar). The TAFII105·NF-κB complex may be required for the positive selection process during lymphocyte development and the immune response by translating upstream positive signals into activation of survival gene products. This idea is supported by our observation of a significant reduction in the relative numbers of single-positive mature T cells in TAFII105ΔC transgenic mice. This effect may be explained in part by the increased rate of apoptosis in the population of immature double-positive thymocytes, from which mature T cells develop by positive selection. However, given that the proportion of the immature double-positive T cells in the thymuses of TAFII105ΔC mice is not decreased despite high apoptotic rates, it is possible that TAFII105 is important for T cell maturation processes that are also apoptosis-independent.The transgenic animals that we have obtained expressed very low amounts of the dominant-negative proteins relative to the native endogenous protein. The accelerated apoptosis of lymphocytes observed despite low expression levels of the TAFII105ΔC transgene may result from the fact that the decision of lymphocytes to undergo apoptosis is dependent on the extent of positive and negative survival signals encountered by the cell. The amount of TAFII105ΔC protein in the lymphocytes of the transgenic animals may be sufficient to shift a delicate balance of pro- and anti-apoptotic signals toward apoptosis. Alternatively, cells expressing higher levels of the transgene are those that may have been eliminated by apoptosis. It is therefore likely that TAFII105 has additional functions that were not explored in this study, as inhibition of other functions of TAFII105 may require expression levels that are at least comparable to those of the native protein. Our inability to obtain transgenic animals expressing high levels of this protein and the decline of its expression over generations raise the possibility that the protein may be toxic to the animals.Early biochemical studies have suggested that activator-TFIID interaction enhances the formation of a functional preinitiation complex (51Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 52Orlando V. Strutt H. Paro R. Methods: A Companion to Methods in Enzymology. 11. Academic Press, Inc., San Diego, CA1997: 205-214Google Scholar, 53Roeder R.G. Trends Biochem. Sci. 1991; 16: 402-408Abstract Full Text PDF PubMed Scopus (346) Google Scholar). A question arising from this study is whether p65/RelA-TAFII105 interaction contributes to the assembly of the preinitiation complex by enhancing recruitment of the substoichiometric TAFII105·TFIID complex to the promoters of anti-apoptotic genes expressed in lymphocytes. Future experiments should address the molecular mechanism by which NF-κB and its coactivators affect the transcription process in vivo. In this study, we have examined the function of TAFII105 in the context of the whole organism and determined the biological importance of a domain in TAFII105 involved in interaction with the NF-κB protein p65/RelA in vitro. We generated and analyzed transgenic mice expressing dominant-negative mutants of TAFII105, which inhibit the function of TAFII105 and, most likely, also of TAFII105 homologs. Because we did not interfere with expression of the native protein, this strategy did not affect the stability of the endogenous TAFII105·TFIID complex. Furthermore, expression of different forms of dominant-negative mutants allowed us to examine the structure-function relationship involved in the mechanism of action of TAFII105 in vivo. We provide evidence that one of the domains within TAFII105 required for interaction with p65/RelA in vitro is crucial for lymphocyte survival and antibody production in the mouse. This notion is consistent with previous findings indicating that TAFII105ΔC-ΔNFκB is a significantly less potent inhibitor of NF-κB transcription activity than TAFII105ΔC (35Yamit-Hezi A. Nir S. Wolstein O. Dikstein R. J. Biol. Chem. 2000; 275: 18180-18187Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and strongly suggests that the C-terminal NF-κB-binding site within TAFII105 is a functionally more important site than the N-terminal site. As NF-κB has been established as a survival factor in lymphocytes (39Barkett M. Gilmore T. Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1066) Google Scholar, 40de Martin R. Schmidt J.A. Hofer-Warbinek R. Mutat. Res. 1999; 437: 231-243Crossref PubMed Scopus (71) Google Scholar, 41Foo S.Y. Nolan G.P. Trends Genet. 1999; 15: 229-235Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 42Grossmann M. O'Reilly L.A. Gugasyan R. Strasser A. Adams J.M. Gerondakis S. EMBO J. 2000; 19: 6351-6360Crossref PubMed Scopus (192) Google Scholar, 43Huesmann M. Scott B. Kieslow P. von Boehmer H. Cell. 1991; 66: 533-542Abstract Full Text PDF PubMed Scopus (321) Google Scholar), the finding that TAFII105 is important for activation of anti-apoptotic genes in B and T lymphocytes implies that it cooperates with p65/RelA in these cells to activate some of the NF-κB target genes. The observation that the NF-κB interaction domain is also required for IgM, IgG2a, and IgG3 isotype switching is also consistent with a recent finding showing that B cells lacking p65 exhibit a defect in switching to IgG3 (44Horwitz B.H. Zelazowski P. Shen Y. Wolcott K.M. Scott M.L. Baltimore D. Snapper C.M. J. Immunol. 1999; 15: 1941-1946Google Scholar). However, it has yet to be determined whether the effect of TAFII105ΔC expression on lymphocyte survival is intrinsic or involves contributions of other cell types. Programmed cell death is of fundamental importance in the immune system and plays important roles in the control of the immune response and in lymphocyte development and cytotoxicity (reviewed in Refs. 45Cohen J.J. Duke R.C. Fadok V.A. Sellins K.S. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1102) Google Scholar, 46Williams G.T. Biochem. Cell Biol. 1994; 72: 447-450Crossref PubMed Scopus (13) Google Scholar, 47Wyllie A.H. Int. Rev. Cytol. 1987; 17: 755-785Google Scholar). In developing lymphocytes, cell death is the mechanism by which immune cells that recognize self-antigens are deleted. This process ensures the release of cells recognizing non-self-antigens into the periphery and determines the finite life span of terminally differentiated cells. Thus, apoptosis provides a flexible mechanism for controlling the composition and size of the mature cell population. It has been proposed that positive selection of thymocytes is a rescue from a default pathway of death in developing thymocytes (48Egerton M. Scollay R. Shortman K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2579-2582Crossref PubMed Scopus (279) Google Scholar, 49Deleted in proofGoogle Scholar, 50Surh C.D. Sprent J. Nature. 1994; 372: 100-103Crossref PubMed Scopus (930) Google Scholar). The TAFII105·NF-κB complex may be required for the positive selection process during lymphocyte development and the immune response by translating upstream positive signals into activation of survival gene products. This idea is supported by our observation of a significant reduction in the relative numbers of single-positive mature T cells in TAFII105ΔC transgenic mice. This effect may be explained in part by the increased rate of apoptosis in the population of immature double-positive thymocytes, from which mature T cells develop by positive selection. However, given that the proportion of the immature double-positive T cells in the thymuses of TAFII105ΔC mice is not decreased despite high apoptotic rates, it is possible that TAFII105 is important for T cell maturation processes that are also apoptosis-independent. The transgenic animals that we have obtained expressed very low amounts of the dominant-negative proteins relative to the native endogenous protein. The accelerated apoptosis of lymphocytes observed despite low expression levels of the TAFII105ΔC transgene may result from the fact that the decision of lymphocytes to undergo apoptosis is dependent on the extent of positive and negative survival signals encountered by the cell. The amount of TAFII105ΔC protein in the lymphocytes of the transgenic animals may be sufficient to shift a delicate balance of pro- and anti-apoptotic signals toward apoptosis. Alternatively, cells expressing higher levels of the transgene are those that may have been eliminated by apoptosis. It is therefore likely that TAFII105 has additional functions that were not explored in this study, as inhibition of other functions of TAFII105 may require expression levels that are at least comparable to those of the native protein. Our inability to obtain transgenic animals expressing high levels of this protein and the decline of its expression over generations raise the possibility that the protein may be toxic to the animals. Early biochemical studies have suggested that activator-TFIID interaction enhances the formation of a functional preinitiation complex (51Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 52Orlando V. Strutt H. Paro R. Methods: A Companion to Methods in Enzymology. 11. Academic Press, Inc., San Diego, CA1997: 205-214Google Scholar, 53Roeder R.G. Trends Biochem. Sci. 1991; 16: 402-408Abstract Full Text PDF PubMed Scopus (346) Google Scholar). A question arising from this study is whether p65/RelA-TAFII105 interaction contributes to the assembly of the preinitiation complex by enhancing recruitment of the substoichiometric TAFII105·TFIID complex to the promoters of anti-apoptotic genes expressed in lymphocytes. Future experiments should address the molecular mechanism by which NF-κB and its coactivators affect the transcription process in vivo. We thank Dr. Ahuva Knyszynski, Dr. Tatyana Burakov, Judith Chermesh, Rafi Saka, and Dr. Ori Brenner (Weizmann Institute of Science Transgenic Facility) for generating and maintaining the transgenic mice and Dr. Sandra Moshonov for editing the manuscript and for technical assistance.